Of all the interventions that have been shown to increase lifespan, dietary restriction (DR) is the most studied and universal aging intervention. Because DR also delays the onset and progression of most age-related diseases as well as maintaining healthspan, it is well accepted that DR increases lifespan by delaying/retarding aging. However, we still do not understand the mechanism(s) responsible for the anti-aging effect of DR. An important aspect of DR that has been largely overlooked is that when implemented early in life DR can increase lifespan of rodents even when rodents are fed ad libitum (AL) the remainder of their life. This provides compelling evidence that DR acts through a mechanism that involves a molecular signal(s) that arises shortly after the implementation of DR and has an impact on the animal over its lifespan, even after DR is discontinued, e.g., an epigenetic mechanism, such as DNA methylation. Functionally, DNA methylation (5mC) can regulate chromatin status and affect the ability of transcription factors and other DNA binding proteins to access DNA and regulate gene expression, thereby altering the function of cells and tissues. Several groups, including our laboratories, have shown that DR alters DNA methylation in tissues of mice, reversing many of the changes in DNA methylation that occur with age as well as inducing changes in DNA methylation that do not change with age. Recently we found that short-term DR induces changes in DNA methylation in intestinal mucosa in the promoter of the Nts 1 gene. The changes in DNA methylation are closely associated with increased expression of Nts 1, which persist when the DR mice are then fed ad libitum for several months. Because the epithelial cells in the intestinal mucosa are continuously renewed every 4 to 5 days, the changes in DNA methylation we observed in intestinal mucosa most likely arise in intestinal stem cells. Therefore, we hypothesize that DR induces changes in DNA methylation in intestinal stem cells at specific genomic sites that results in a molecular memory, which potentially leads to alterations in the expression of genes that are important in intestinal stem cell function. In this project, we will measure changes in DNA methylation (5mC) and hydroxymethylation (5hmC) induced by DR in intestinal stem cells using a novel assay developed by our group, which allows us to measure accurately at single base resolution changes in both 5mC and 5hmC at ~ 30 million specific sites in the genome. In this R21 grant, we will test our hypothesis in the following specific aims. Aim 1: Determine the effect of DR on DNA methylation and hydroxymethylation across gene regulatory regions of the genome of stem cells isolated from the intestines of three groups of mice: mice fed AL, mice fed DR for 4 months, and DR mice fed DR for 4 months and then fed AL for 6 months. Aim 2: Identify changes in transcription that could arise from changes in DNA methylation/hydroxymethylation and play a role in intestinal stem cell function.